Metabolic dysregulation linked to sickling tendency in sickle cell anemia independent of HbF

Sickle cell disease (SCD) presents substantial interindividual clinical variability that remains only partially explained by genotype, fetal hemoglobin (HbF), and co-inherited alpha-thalassemia. We investigated whether endogenous metabolites modulate the sickling tendency, independently of HbF. We analyzed 368 steady-state HbSS patients with oxygen gradient ektacytometry (Point of Sickling [PoS]) and untargeted metabolomics (DI-HRMS, 1309 metabolites). PoS was adjusted for HbF for ANOVA analysis (A-FDR p ≤ 0.05). Linear regression (lr) models were adjusted for HbF, age, alpha-thalassemia, Oxygenscan device, and hydroxyurea (lr-FDR p ≤ 0.05). 111 metabolites differed between adjusted-PoS groups, and 27 metabolites associated with PoS in lr. Seven metabolites overlapped across approaches. Increased sickling (higher PoS) positively correlated with glycolytic and pentose phosphate pathway intermediates, glyceraldehyde-3-phosphate (A-FDR <0.001), 2,3-DPG (A-FDR = 0.002; lr-FDR = 0.004), and ribulose-5-phosphate (A-FDR <0.001). RBC membrane remodeling markers, including carnitines (lr-FDR = 0.002), and oxidative stress and inflammation metabolites: glutathione disulfides (A-FDR = 0.001), acetylated citrulline (A-FDR < 0.001; lr-FDR = 0.009), spermidine (lr-FDR = 0.03), and acetylated tryptophan (A-FDR = 0.03; lr-FDR = 0.03), were also elevated in this group. Low PoS associated with downstream tryptophan metabolites (A-FDR < 0.001, 0.006), indicating inflammatory regulation. Overall, this study demonstrates that metabolic processes are associated with sickling tendencies independent of HbF, providing insights into SCD heterogeneity, identifying candidate metabolic pathways, and supporting current strategies to target glycolysis in SCD.